Animal (2010), 4:5, pp 739–744 & The Animal Consortium 2010 animal doi:10.1017/S1751731109991728 Effects of inulin and lactulose on the intestinal morphology of calves - S. Masanetz1 , N. Wimmer1, C. Plitzner2a, E. Limbeck1, W. Preißinger3 and M. W. Pfaffl1 1Chair of Physiology, Research Center for Nutrition and Food Sciences (ZIEL), Technical University of Munich, D-85354 Freising, Germany; 2University of Natural Resources and Applied Life Sciences, Division of Animal Food and Nutrition, A-1180 Vienna, Austria; 3Bavarian State Research Centre for Agriculture (LfL), D-85586 Poing-Grub, Germany (Received 10 February 2009; Accepted 12 December 2009; First published online 13 January 2010) For some time now prebiotics have been proposed to improve health by stimulation of beneficial bacteria in the intestine of humans and animals. The current study is aiming to show effects of feeding of either 2% inulin or 2% lactulose in milk replacer on performance and intestinal morphology of male Holstein–Friesian calves. After 20 weeks of feeding inulin led to significantly higher daily weight gains than lactulose while control animals ranged between the experimental feedings. Ingestion of milk replacer was reduced in lactulose treated animals. Additionally differences of villus height in jejunum ( P 5 0.07) and ileum ( P 5 0.03) could be found with an increase for lactulose treated animals and a decrease for inulin treated animals. In ileum the density of proliferative epithelial cells tended to be lower in inulin treated and higher in lactulose treated animals ( P 5 0.08). Both inulin and lactulose tended to decrease the quantity of goblet cells in the tips of ileal villi ( P 5 0.07). Both prebiotics can affect performance and intestinal morphology of calves and may as such affect animal health. But effects differ between substances. Keywords: calves, intestine, inulin, lactulose, morphology, prebiotics Implications bacterial populations towards a healthier flora (Gibson and Roberfroid, 1995). Research on prebiotic substances has been done mostly on Inulin is a natural constituent of a wide range of plants human subjects or small laboratory animals. And mostly including many common vegetables and cereals in western only changes in gut bacteria populations were investigated diet (Van Loo et al., 1995). It consists of b-(2-1)-linked disregarding effects on the intestine itself. Since prebiotics fructo-oligosaccharides of varying degrees of polymeriza- are claimed to be a possible alternative to antibiotics as tion from 2 to 60 sugar units. In the intestine inulin leads to growth promoters in livestock husbandry this study has a shift in the bacterial flora towards more bifidobacteria been done on calves to further investigate potential bene- regarded as beneficial for host health (Gibson et al., 1995). ficial effects on cattle health. To determine modes of action Additionally inulin-type fructans have been shown to of the prebiotics inulin and lactulose changes of morphol- have anti-carcinogenic properties (Hughes and Rowland, ogy in the small and large intestine and the adjoining 2001; Femia et al., 2002). Fructo-oligosaccharides were also immune defense tissues have been observed. reported to enhance the performance of livestock including pigs and calves (reviewed by Van Loo, 2007). Introduction Lactulose (4-O-b-D-galactopyranosyl-D-fructose) is a semi-synthetically produced disaccharide that does not In recent years the European Union introduced a ban on occur naturally (Schumann, 2002). It is fermented by lac- antibiotics as growth promoters in animal feed. Now tobacilli and bifidobacteria, but also by other bacteria alternatives have to be found to assure animal health and species such as Clostridium perfringens, Escherichia coli or performance during meat or milk production. Possible Bacteroides spp. (Mitsuoka et al., 1987). Lactulose is com- beneficial feed additives may be prebiotics such as inulin monly used to treat constipation (Attar et al., 1999) and or lactulose which can be able to modulate intestinal hepatic encephalopathy (Bircher et al., 1966) but also to a enhance the animal performance (Fleige et al., 2007). Present address: Vereinigung der Su¨ dtiroler Tierzuchtverba¨nde, I-39100 Bolzano, Italy. Until now most of the studies were done on human - E-mail: [email protected] subjects or laboratory rats instead of livestock and mostly 739 Masanetz, Wimmer, Plitzner, Limbeck, Preißinger and Pfaffl studied changes in bacterial flora. Comparably little research parts of jejunum, ileum and colon as well as mesenteric has been done on possible changes of intestinal morphology. lymphoid nodes were collected. Tissue samples were washed Thus the object of this study was to provide information about in physiological NaCl solution and placed in neutral buffered of the influences of inulin and lactulose feeding on performance, 3.7% formalin (Carl Roth GmbH, Karlsruhe, Germany) for 24 h. morphology of the intestinal mucosa and on mesenteric At the Landesuntersuchungsamt (LUA, Oberschleißheim) lymphoid nodes in pre-ruminant calves. sampleswhereembeddedinparaffin.Sectionsof4to6mm were cut (Microtom LEICA RM2145, Leica, Wetzlar, Germany), deparaffinized and rehydrated before further treatment. All Material and methods microscopic analyses were done randomly by one person without knowledge of treatment groups. Histological sections Animals, husbandry, feeding and experimental procedures of ileum, jejunum and mesenteric lymphoid nodes were Forty-two male Holstein–Friesian calves were purchased examined with the light microscope Axioskop 2 plus (Zeiss, from the Viehzentrum Waldkraiburg GmbH and housed at Oberkochen, Germany), sections of the colon with the stereo- the experimental station Karolinenfeld (Bayerische Land- microscope Stemi 2000-C (Zeiss). Pictures were taken with the esanstalt fu¨ r Landwirtschaft – LfL, Institut fu¨ r Tiererna¨hrung AxioCam MRc (Zeiss) and analyzed with the connected soft- und Futterwirtschaft). The animals were subdivided into ware AxioVision 3.1. three experimental groups (n 5 14 per group) with balanced weight (52.9 6 6.2 kg) and age (22 6 5 days). Compositions of the three diets are given in Table 1. Calves Histomorphometry of the control group were fed with the milk replacer Milkibeef Histomorphometry was done on sections stained with Alcian Top (Milkivit, Trouw Nutrition, Burgheim, Germany). The other blue/periodic acid Schiff’s reagent (AB-PAS) (see histochem- groups were fed with the same milk replacer iso-energetically istry) or haematoxylin (Carl Roth GmbH, Karlsruhe, Germany) andiso-nitrogenicallyenrichedwitheither2%inulin(Beneo R and eosin yellowish solution (Fluka-Chemie AG, Buchs, Swit- ST, Orafti, Tienen, Belgium) or 2% lactulose (Lactusat, Milei zerland). Measurement techniques were adapted from Sehm GmbH, Leutkirch, Germany). Calves were fed individually et al. (2006). Briefly villus length from lamina muscularis to tip by transponder automatic feeders (Fo¨rster Technik, Engen, and width were measured in sections of jejunum and ileum. Germany). During the experimental period the milk replacer Crypt depth from lamina muscularis to the crypt mouth and concentration was rising from 125 g/l to 200 g/l with daily distance between crypts were examined in sections of the intake volumes rising from 6 l to 16 l. Calves had free group colon. Figure 1 gives an overview of conducted measurements accesstofreshdrinkingwaterandupto300ghayperday on the intestinal mucosa. All were done on three well-defined and animal. Since the calves were housed on straw a further villi or crypts of one section. In mesenteric lymphoid nodes and uptake of roughage could not be excluded. The animals were in ileal Peyer’s patches the number of lymph follicles in a slaughtered after 20 weeks. defined area and areas of these follicles were measured. Histological sampling Histochemistry Immediately after slaughtering the gastrointestinal tract was The AB-PAS staining method was used to investigate the removed from the carcass. Sections of 0.5 to 1 cm of centre number of goblet cells in jejunum and ileum. Briefly sections Table 1 Ingredients and analysis of nutrient and energy content of the diets (energy content of the milk replacer was estimated with the program Zifo (LfL, 2005)) Control Inulin Lactulose Hay Straw 50% of fat concentrate (50% of whey powder, 50% of coconut/palm oil) 38.3 38.3 38.5 Skimmed milk (%) 50.2 50.2 50.2 Pregelatinised wheat starch (%) 4.4 2.8 3.5 Whey protein concentrate (%) 4.1 3.5 – Beneo R ST (%) – 2.2 – Lactusat (%) – – 4.8 Vitamins, minerals, amino acid mix (%) 2.0 2.0 2.0 80% of soybean oil/20% of emulsifier (%) 0.985 0.985 0.985 Aroma (%) 0.015 0.015 0.015 DM (g/kg) 963 959 960 880 892 Crude ash (g/kg DM) 73 73 70 39 32 Crude protein (g/kg DM) 225 229 230 112 35 Ether extracts (g/kg DM) 210 208 211 15 13 Crude fiber (g/kg DM) 6 2 4 323 460 Energy (MJ/kg DM) 16.9 16.9 17.0 9.46 6.9 DM 5 dry matter. 740 Effects of prebiotics on the intestine of calves Figure 1 Morphological measurements in the small and large intestine: Conducted measurements are shown as an overview for both small and large intestine (B) and as actual measurements in sections of jejunum (A) and colon (C). (1) lamina muscularis mucosae, (2) crypt of Lieberku¨ hn, (3) villus; small intestine: (a) villus length, (b) villus width, (c) villus tip; large intestine (no villi existent), (d) crypt depth, (e) distance between crypts. hydrogen peroxide and 10% goat serum (Dako cytomation), respectively. Binding of MIB-1 (1 : 50 in PBS) was done at 48C over night. Afterwards sections were incubated with horse raddish peroxidase-labeled polyclonal goat anti-mouse antibody (1 : 50 in PBS, Dako cytomation). Visualization was done with 3,30-diaminobenzidine solution (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). Tissue was then counterstained with haematoxylin. Quantifica- tion of MIB-1 positive cells was done similarly to AB-PAS positive cells in the crypts of jejunum, ileum and colon. Statistical analysis For each parameter the mean group values and the residual standard error of the mean were determined.